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Oi:10.1371/journal.pone.0053489.gThe Amount of Added pHE, S, Needs Not to Be Highly AccurateN0 of the internal reference gene was estimated through fluorospectrometric quantification. This is highly recommended as the quantification obtained from another commonly used Table 2. Effects of pHE DNA addition on PCR efficiency.technique, UV spectrometric analysis, is not accurate due to severe interference from impurities commonly present in plant DNA samples. Sometimes, even with a fluorescence-based spectrometric technique, the quantification result may also not be highly accurate because of interference from impurities. This can also be the case for the standard DNA added.SampleaVolume of tomato genomic DNA (ml)bVolume of pHE (ml)cELIPAverage 5 level a a a 1 level A A AHPTAverage 1.87660.041 1.83960.018 1.88060.024 5 level a a a 1 level A A AA B Ca1 10 11.88760.036 1.87560.026 1.86560.A two-copy transgenic T0 tomato plant was used; The concentration was 10.20 ng ml21, containing 10,000 ELIP and 10,000 HPT molecules ml21; The concentration was 0.051 pg ml21, containing 10,000 ELIP and 10,000 HPT molecules ml21. doi:10.1371/journal.pone.0053489.tb cA 25331948 qPCR Approach for Fruquintinib biological activity transgene Copy Number AnalysisFigure 3. Calculation of N0ELIP when taking a Cb within Ib 210 to Ib +5. The values above/beneath the arrows indicate the coefficient of variation. Line 1-Line 6 (L1-L6) were the six transgenic T0 tomato lines; Ib was set as the integer part for Ctb; Cb indicated cycles in the exponential amplification phase of sample B. doi:10.1371/journal.pone.0053489.gHowever, with a recombinant plasmid harboring both r and t served as the standard DNA, the amount added to the test samples does not need to be highly accurate. As clearly shown in Equation (20), the transgene copy number result was independent of S, the amount of added standard DNA.Transgene Copy Number MedChemExpress 11089-65-9 determination of Six Transgenic Tomato PlantsThe HPT gene copy numbers in transgenic tomato plants were determined by the ratio of N0HPT to N0ELIP. The transgene copy number should be twice the ratio because of the diploid nature of tomato. In the present study, samples from six individual T0 plants were analyzed: the data indicated that three lines had one copy of the transgene, while the other three lines had two copies (Table 3). The transgene copy number of these six transgenic tomato plants was also analyzed by Southern blot (Figure 4), which confirmed the SAQPCR results obtained in the present study.Table 3. Determination of transgene copy number of six transgenic tomato plants by standard addition qPCR (SAQPCR).Line 1 2 3 4 5N0HPT 4476.61 4719.62 4699.55 9386.66 9672.54 9635.N0ELIP 9273.31 9912.54 9084.33 9361.90 9901.20 9565.N0HPT/N0ELIP 0.48 0.48 0.51 1.00 0.97 1.Transgene copy numbera 1 1 1 2 2Prospects for the Application of SAQPCR Approach to other Plants and OrganismsThe transgene copy number of six transgenic tomato plants obtained by the SAQPCR approach was confirmed by Southern blot analysis (compare results in Table 3 and Figure 4). However, unlike the latter, only a small amount of plant tissue is required for SAQPCR, and the analysis can be completed in a much shorter time. Therefore, the approach is especially suitable for high throughput transgene copy number determination in small-sized plant species or cultivars, as well as slow-growing woody plants. Although the approach was established on tomato with singlecopy ELIP as the r and HPT as t, it is generally suitable for o.Oi:10.1371/journal.pone.0053489.gThe Amount of Added pHE, S, Needs Not to Be Highly AccurateN0 of the internal reference gene was estimated through fluorospectrometric quantification. This is highly recommended as the quantification obtained from another commonly used Table 2. Effects of pHE DNA addition on PCR efficiency.technique, UV spectrometric analysis, is not accurate due to severe interference from impurities commonly present in plant DNA samples. Sometimes, even with a fluorescence-based spectrometric technique, the quantification result may also not be highly accurate because of interference from impurities. This can also be the case for the standard DNA added.SampleaVolume of tomato genomic DNA (ml)bVolume of pHE (ml)cELIPAverage 5 level a a a 1 level A A AHPTAverage 1.87660.041 1.83960.018 1.88060.024 5 level a a a 1 level A A AA B Ca1 10 11.88760.036 1.87560.026 1.86560.A two-copy transgenic T0 tomato plant was used; The concentration was 10.20 ng ml21, containing 10,000 ELIP and 10,000 HPT molecules ml21; The concentration was 0.051 pg ml21, containing 10,000 ELIP and 10,000 HPT molecules ml21. doi:10.1371/journal.pone.0053489.tb cA 25331948 qPCR Approach for Transgene Copy Number AnalysisFigure 3. Calculation of N0ELIP when taking a Cb within Ib 210 to Ib +5. The values above/beneath the arrows indicate the coefficient of variation. Line 1-Line 6 (L1-L6) were the six transgenic T0 tomato lines; Ib was set as the integer part for Ctb; Cb indicated cycles in the exponential amplification phase of sample B. doi:10.1371/journal.pone.0053489.gHowever, with a recombinant plasmid harboring both r and t served as the standard DNA, the amount added to the test samples does not need to be highly accurate. As clearly shown in Equation (20), the transgene copy number result was independent of S, the amount of added standard DNA.Transgene Copy Number Determination of Six Transgenic Tomato PlantsThe HPT gene copy numbers in transgenic tomato plants were determined by the ratio of N0HPT to N0ELIP. The transgene copy number should be twice the ratio because of the diploid nature of tomato. In the present study, samples from six individual T0 plants were analyzed: the data indicated that three lines had one copy of the transgene, while the other three lines had two copies (Table 3). The transgene copy number of these six transgenic tomato plants was also analyzed by Southern blot (Figure 4), which confirmed the SAQPCR results obtained in the present study.Table 3. Determination of transgene copy number of six transgenic tomato plants by standard addition qPCR (SAQPCR).Line 1 2 3 4 5N0HPT 4476.61 4719.62 4699.55 9386.66 9672.54 9635.N0ELIP 9273.31 9912.54 9084.33 9361.90 9901.20 9565.N0HPT/N0ELIP 0.48 0.48 0.51 1.00 0.97 1.Transgene copy numbera 1 1 1 2 2Prospects for the Application of SAQPCR Approach to other Plants and OrganismsThe transgene copy number of six transgenic tomato plants obtained by the SAQPCR approach was confirmed by Southern blot analysis (compare results in Table 3 and Figure 4). However, unlike the latter, only a small amount of plant tissue is required for SAQPCR, and the analysis can be completed in a much shorter time. Therefore, the approach is especially suitable for high throughput transgene copy number determination in small-sized plant species or cultivars, as well as slow-growing woody plants. Although the approach was established on tomato with singlecopy ELIP as the r and HPT as t, it is generally suitable for o.